The Expression Difference And Function Of Two CENP-E MRNA Forms In Different Tumor Tissues

CENP-E is an important spindle assemble checkpoint (SAC) protein. SAC maintains the stability of chromosome number by monitoring chromosome segregation[1], and has an important role in cell cycle and tumorigenesis. There are two different forms of CENP-E mRNA in Genebank database, CENP-EWT and CENP-Eâ… , and CENP-Eâ… misses the 38th exon. HeLa cells were reported to express CENP-Eâ… [2], but our group found there was CENP-EWT without lacking the 38th exon in previous experiments. Therefore, whether CENP-EWT and CENP-Eâ… exist in the same cells or not, which form is the dominant, whether the presence of them are different in tumor cells and normal cells or not, whether there are deletions at DNA level or not, whether their functions are different or not, the solvation of these problems will help us to further study CENP-E in the spindle checkpoint function.In this study, we detected different types of form and expression of CENP-E at DNA and mRNA levels in different cell lines and cancer tissues and adjacent tissues by nested PCR, for the first time, we found the two mRNA forms(CENP-EWT and CENP-Eâ… ) existed in the same cell, CENP-Eâ… had some relevance with the malignancy of cells, but there is no loss at the DNA level. Then, we successfully constructed two interference plasmids for two CENP-E mRNA forms, the results showed that the CENP-EWT disturbance can increase the malignancy of cells, thus provided new idea to further study the possible role of CENP-E in carcinogenesis and clinical treatment of tumor.PARTâ… THE EXPRESSION DIFFERENCE OF TWO CENP-E mRNA FORMS IN DIFFERENT TUMOR TISUESSObjective:1. Analyzing the expression of two CENP-E mRNA forms in Hela cells.2. Analyzing the expression of two CENP-E mRNA forms in 13 cell lines (U251, HepG2, LO2, 786-0, OS-RC-2, HEK293, BIU-87, T24, SW480, HCT116, A549, MDA-MB-231 and MCF7) and 9 types of cancer tissues (hepatocellular carcinoma, nasopharyngeal carcinoma, suprarenal epithelioma, bladder carcinoma, colon carcinoma, lung cancer, breast carcinoma, gastric carcinoma and esophageal cancer) and corresponding adjacent tissues.3. Determining whether there is deletion of the 38th extron in genomic DNA or not.Methods:1. Amplifying the cDNA of CENP-E from the total RNA of Hela cells by RT-PCR method to analysis the expression of two CENP-E mRNA forms.2. Simultaneous amplifying two CENP-E mRNA using nested RT-PCR to analysis weather there is difference between their expression in different cell lines and in cancer tissues and corresponding adjacent tissues or not, and weather the difference is statistically significant.3. Amplifying genomic DNA by nested PCR in different cell lines and cancer tissues and corresponding adjacent tissue to analysis weather the exon 38 deletion exist at the DNA level.Results:1. Two CENP-E mRNA forms are exist simultaneously in Hela cells.2. There is difference between their expression of Two CENP-E mRNA forms in different cell lines and in cancer tissues and corresponding adjacent tissues, and the difference is statistically significant.3. The exon 38 deletion do not exist at the DNA level in different cell lines and in cancer tissues and corresponding adjacent tissues, and the difference is not statistically significant.Conclusion: The two forms of CENP-E mRNA exist in the same cell, The deletion of the 38th exon presents at the RNA level rather than at the DNA level, CENP-Eâ… may be associated with the tumor.PARTâ…¡Construct and screen interference plasmids for the CENP-EWTObjective:1. Constructing recombinant plasmid vectors expressing short hairpin RNA (shRNA) targeting at CENP-EWT.2. Screening plasmids that can effectively interfere CENP-EWT and the best transfection system.Methods:1. ShRNA sequences targeting at CENP-EWT were designed and synthesized, and then ligated with vector pgenesil-1 to construct shRNA recombinant plasmids.2. NK293 cells transfected by shRNA plasmids were detected the expression changes of CENP-EWT using PCR to screen effective interference plasmid and the best transfection system.Results: Effective shRNA plasmids against CENP-EWT and the best transfection system are successful designed, synthesized and constructed, screened. They can inhibit the expression of target gene in the NK293 cells, and has laid a solid foundation for further study. PART IIIInfluence to cells after Interfereing the two forms of CENP-E mRNAObjective:1. Exploring the influence to proliferation of HepG2 and LO2 cells after interference with the two forms of CENP-E mRNA.2. Exploring the influence to cell cycle of HepG2 and LO2 cells after interference with the two forms of CENP-E mRNA.3. Exploring the influence to chromosome segregation of HepG2 and LO2 cells after interference with the two forms of CENP-E mRNA.Methods:1. MTT experiment was used to prepare the cell growth curve of cells before and after RNAi, which could be used to evaluate the influence of cell growth by gene inhibition.2. The influences of the two genes on cell cycle were evaluated by FCM.3. Analysis of mitotic index and chromosome karyotype of cells before and after RNAi was applied to detect the influence of intergenic suppression to chromosomal separation.Results:1. MTT showed the experimental group growed significantly slower than the control group after the effective interference with CENP-EWT, this phenomenon was more pronounced after the two forms of CENP-E mRNA were interferenced.2. MTT showed the cells in G2 / M phase increased after the effective interference with CENP-EWT, this phenomenon was more pronounced after the two forms of CENP-E mRNA were interfered.3. Mitotic index determination showed the experimental group increased the proportion of dividing cells; karyotype analysis showed that the proportion of chromosome abnormal cells in the experimental group increased significantly. This phenomenon was more pronounced after the two forms of CENP-E mRNA were interfered.Conclusion: Cells grew slowly, mitotic cells increased and ratio of the cells with abnormal chromosomal number was increased after the form of CENP-EWT were interfered, which demonstrated CENP-EWT and CENP-E might play an important regulatory role in cell proliferation and chromosome separation. We successfully constructed a cell model in vitro to observe abnormal changes in chromosome number after CENP-EWT was inhibited which established the foundation for the later stage of clinical research.